Chromosome fragile sites have diverse structures, yet there is no clear correlation between the structure of a fragile site and its stability. It is therefore difficult to devise a simple explanation for fragile site instability, and this gap in knowledge prevents us both from understanding the mechanism by which these sequences alter chromosome structure and also from developing therapeutic interventions to preserve genome integrity. The objective of this application is to use yeast as a model system to understand how the arrangement of retrotransposon DNA can create fragile sites and alter the propensity of a DNA locus to undergo rearrangement. A pair of inverted retrotransposons on yeast chromosome III suffers an increased incidence of double strand breaks and chromosome translocations, suggesting that repetitive sequences that are present in tandem or inverted orientation function as fragile sites. The central hypothesis for the proposed research is that repetitive DNA content and organization will be a crucial factor in determining the frequency with which genetic loci display instability. The central hypothesis will be tested by using an isogenic series of retrotransposon overdose (RO) strains that contain an elevated number of retrotransposons dispersed throughout the genome, including numerous pairs of retrotransposons that are potential fragile sites; these strains are a unique genetic system that enable the study multiple fragile sites within the same genome. The contribution of fragile site structure to genome instability will be investigated by pursuing the following specific aims: (1) identifying genomic loci that are most prone to instability following replication stress in RO strains, and (2) determining the relative stability of predicted fragile sites following lab evoluton. This innovative approach is expected to produce the following outcomes. First, a more viable model for human fragile sites will be developed. This RO model system can represent the diversity in sequence and structure of human fragile sites, thereby elucidating why human fragile sites differ dramatically in their stability. Second, the evolutionary stability of yeast fagile sites will be determined, thereby elucidating why fragile sites are maintained during evolution and whether they correlate with conserved chromosome breakpoints. The outcome of the proposed studies will be a more thorough understanding of the relationship between DNA structure and genome stability in yeast, a prerequisite to understanding the mechanism of chromosome fragility. PUBLIC HEALTH RELEVANCE: Fragile sites are chromosome regions that are prone to instability, and chromosome rearrangements that may result from this increased incidence of DNA breakage represent some of the earliest changes in cancer progression. This study will employ a novel humanized yeast model system that contains fragile sites with diverse structures. The proposed research is relevant to public health because it will allow us to understand why fragile sites differ dramatically in their stability, thereby allowing us to monitoror target specific fragile sites to prevent genome instability.